More than 8 million organic compounds are known and many are thought to be biodegradable by microorganisms, the principle agents for recycling organic matter on Earth. In this context, microbial enzymes represent the greatest diversity of novel catalysts. This is why microbial enzymes are predominant in industrial enzyme technology and in bioremediation, whether used as purified enzymes or in whole cell systems.
Effects of s-triazine Herbicide Use
Modern agricultural practices rely heavily on the use of herbicides to control weed populations. S-triazine (i.e., symetric triazine) herbicides, primarily atrazine and simazine, are widely used herbicides for selective control of broadleaf weeds and some grasses in a variety of crops. Since atrazine and other s-triazine herbicides biodegrade relatively slowly in soils, label directions for the use of atrazine restrict the types of crops that can be planted to prevent carryover problems in the next growing season. For example, alfalfa and soybeans are susceptible to atrazine concentrations in soil ranging from 0.09 mg/Kg to 0.53 mg/Kg, depending on the concentration of soil organic matter.
Numerous studies on the environmental fate of atrazine have shown that atrazine is a moderately persistant compound that is transformed to CO2 very slowly, if at all, under aerobic or anaerobic conditions. It has a water solubility of 33 mg/l at 27xc2x0 C. Its half-life (i.e., time required for half of the original concentration to dissipate) can vary from about 4 weeks to about 57 weeks when present at a low concentration (i.e., less than about 2 parts per million (ppm)) in soil. High concentrations of atrazine, such as those occurring in spill sites have been reported to dissipate even more slowly.
As a result of its widespread use, atrazine is sometimes detected in water in concentrations exceeding the maximum contaminant level (MCL) of 3 xcexcg/l (i.e., 3 parts per billion (ppb)), a regulatory level that took effect in 1992. Point source spills of atrazine have resulted in levels as high as 25 ppb in some wells. Levels of up to 40,000 mg/l (i.e., 40,000 ppm) atrazine have been found in the soil at spill sites more than ten years after the spill incident. Point source spills and subsequent runoff can result in the presence of atrazine in surface, subsurface, and ground water.
While earlier studies have reported atrazine degradation only by mixed microbial consortia, more recent reports have indicated that several isolated bacterial strains can degrade atrazine. In fact, research groups have identified atrazine-degrading bacteria classified in different genera, including Rhodococcus sp. and Pseudomonas sp., from several different locations in the U.S. (e.g., Minnesota, Iowa, Louisiana, and Ohio) and Switzerland (Basel).
An atrazine-degrading bacterial culture, identified as Pseudomonas sp. strain ADP, ATCC No. 55464, was isolated and was found to degrade atrazine at concentrations greater than about 1,000 xcexcg/ml under growth and non-growth conditions. See Mandelbaum, et al. (U.S. Pat. No. 5,508,193). Pseudomonas sp. strain ADP (Atrazine Degrading Pseudomonas) uses atrazine as a sole source of nitrogen for growth. The organism completely mineralizes the s-triazine ring of atrazine under aerobic growth conditions. That is, this bacteria is capable of degrading the s-triazine ring and mineralizing organic intermediates to inorganic compounds and/or ions (e.g., CO2 and NH4).
Herbicide Resistant Plants
More than 35 species of plants have been reported to be naturally resistant to s-triazine herbicides. Typically, these plants degrade atrazine via glutathione s-transferase reactions (i.e., the atrazine is conjugated to glutathione and subsequently degraded). Alternatively, these plants alter the protein atrazine binds, quinone-binding (QB) protein, which is a component of photosystem II in the chloroplast. Atrazine resistant weeds have been reported to have an altered QB protein which has a 1000-fold reduced affinity for atrazine; however, these plants typically do not compete well in natural systems due to decreased photosynthesis efficiency. Furthermore, these plants do not degrade s-triazines. In the case of atrazine resistant plants, cross resistance to other s-triazine herbicides appears to be relatively common (Ottmeier, W. et al., Pesticide Biochem. Physiol., 18, 357-367. (1982)). Thus, there is a need for methods to remove s-triazines from the environment.
In view of the occasional prevalence of s-triazines in the environment at levels above regulatory standards, there is a need in the art for methods to remediate, i.e., remove, s-triazines present in the environment, including soil and water. Thus, preferred aspects of the present invention provide transgenic plants that are resistant to s-triazine compounds, and methods of making and using such plants. Preferably these plants will degrade s-triazines, more preferably detoxify s-triazines, to more quickly reduce the occurrence of s-triazines in soil and water.
In a preferred embodiment, the present invention provides transgenic alfalfa plants that express a bacterial atrazine chlorohydrolase enzyme, AtzA. AtzA converts atrazine to hydroxyatrazine, which appears to have no herbicidal activity and which is relatively immobile in soil. Alfalfa has rooting characteristics which allow it to explore shallow and deep soils, thus it provides for the remediation of contaminated soils and possibility of remediating contaminated surface and subsurface water.
The present invention provides a transgenic plant including an exogenous coding region encoding an enzyme that imparts resistance to, and optionally degrades, at least one s-triazine. The s-triazine can be atrazine. The enzyme can dehalogenate at least one s-triazine, and if the s-triazine is atrazine, the atrazine can be converted to hydroxyatrazine.
The nucleotide sequence of the exogenous coding region can be nucleotides 58-1480 of SEQ ID NO:1. Alternatively, the complement of the nucleotide sequence of the exogenous coding region hybridizes to the nucleotide sequence set forth at nucleotides 58-1480 of SEQ ID NO:1 in a solution containing 250 mM Na2HPO4, pH 7.4, 2 ml/liter 0.5 M EDTA, pH 8.0, and 10 grams/liter bovine serum albumin at 65xc2x0 C. for at least 4 hours, followed by three washes for twenty minutes each at 65xc2x0 C. in a solution containing 2xc3x97SSC and 0.1% SDS.
The invention includes seeds of the transgenic plant, the progeny of a first or subsequent generation of the transgenic plant and the seeds thereof, and the seeds of the progeny of the first or subsequent generation of the transgenic plant. The plant can be a dicot, including an alfalfa plant, or a monocot, including a grass. A hybrid plant resistant to at least one s-triazine and an inbred plant resistant to at least one s-triazine, prepared from the transgenic plant, is also included in the present invention. In the transgenic plants, the exogenous coding region can impart resistance to levels of at least one triazine that inhibit the growth of a nontransgenic plant.
The invention is also directed at a method for degrading at least one s-triazine, including planting a plant in a composition containing an s-triazine wherein the plant degrades, and optionally detoxifies, at least one s-triazine in the composition, and growing the plant in the composition so that the plant degrades, and optionally detoxifies, at least one s-triazine. The plant can include an exogenous coding region that produces an enzyme capable of degrading, and optionally detoxifying, at least one s-triazine. The composition can include soil and/or water, and the plant can decrease the concentration of at least one s-triazine in the soil and/or the water. The at least one s-triazine can be selected from the group of atrazine, desethylatrazine, deisopropylatrazine, desethylhydroxyatrazine, desisopropylhydroxyatrazine, desethyldesisopropylatrazine, simazine, terbuthylazine, melamine, ammelide, ammeline, prometryn, ametryn, and propazine. The plant can be a dicot, including an alfalfa plant.
Another aspect of the invention is a method of imparting to a plant resistance to at least one s-triazine, including transforming a cell of a susceptible plant with a nucleic acid fragment including an exogenous coding region encoding an enzyme that degrades, and optionally detoxifies, at least one s-triazine, regenerating the transformed plant cell to provide a plant, and identifying a transformed plant which expresses the coding region so as to render the plant resistant to at least one s-triazine. The plant can be a dicot, including an alfalfa plant.
Definitions
xe2x80x9cNucleic acid fragmentxe2x80x9d as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA (both genomic and plasmid) and both double- and single-stranded RNA. A polynucleotide fragment may include both coding and non-coding regions that can be obtained directly from a natural source (e.g., a microorganism), or can be prepared with the aid of recombinant or synthetic techniques. A xe2x80x9cnucleic acid moleculexe2x80x9d may be equivalent to this nucleic acid fragment or it can include this fragment in addition to one or more other nucleotides. For example, a nucleic acid molecule of the invention can be a vector, such as an expression or cloning vector.
xe2x80x9cPolypeptidexe2x80x9d as used herein refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like.
xe2x80x9cHost cellsxe2x80x9d refer to, for example, microorganisms, including prokaryotic (Eubacteria and Archea) microorganisms (e.g., E. coli and cyanobacteria), and eukaryotic microorganisms (e.g., Chlamydomonas) and plant cells that can be used as recipients for introduction of a vector.
xe2x80x9cCoding regionxe2x80x9d refers to a nucleic acid fragment that encodes a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of the coding region are generally determined by a translation start codon at its 5xe2x80x2 end and a translation stop codon at its 3xe2x80x2 end. xe2x80x9cExogenous coding regionxe2x80x9d refers to a foreign coding region, i.e., a coding region that is not normally present in a host cell. An exogenous coding region of the present invention typically contains no introns, but can be altered by methods known to the art of molecular biology to contain introns that are not present in the wild-type coding region. A coding region can be linked to a nucleic acid fragment encoding a transit or signal peptide, for instance a chloroplast transit peptide, that causes the polypeptide encoded by the coding sequence to be targeted to a particular compartment of a host cell.
xe2x80x9cRegulatory regionxe2x80x9d refers to a nucleic acid fragment that regulates expression of a coding region to which a regulatory region is operably linked. Non-limiting examples of regulatory regions include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and terminators.
xe2x80x9cOperably linkedxe2x80x9d refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulatory element is xe2x80x9coperably linkedxe2x80x9d to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory region.
xe2x80x9cTransformationxe2x80x9d refers to the introduction of a nucleic acid fragment into a host cell, irrespective of the method used for introduction. An introduced nucleic acid fragment may be integrated into the host genomic DNA, or alternatively, maintained as a non-integrated vector, for example, as a plasmid.
The term xe2x80x9ccomplementxe2x80x9d and xe2x80x9ccomplementaryxe2x80x9d as used herein, refers to the ability of two single stranded nucleic acid fragments to base pair with each other, where an adenine on one nucleic acid fragment will base pair to a thymine on a second nucleic acid fragment and a cytosine on one nucleic acid fragment will base pair to a guanine on a second nucleic acid fragment. Two nucleic acid fragments are complementary to each other when a nucleotide sequence in one nucleic acid fragment can base pair with a nucleotide sequence in a second nucleic acid fragment. For instance, 5xe2x80x2-ATGC and 5xe2x80x2-GCAT are complementary. The terms complement and complementary also encompass two nucleic acid fragments where one nucleic acid fragment contains at least one nucleotide that will not base pair to at least one nucleotide present on a second nucleic acid fragment. For instance the third nucleotide of each of the two nucleic acid fragments 5xe2x80x2-ATTGC and 5xe2x80x2-GCTAT will not base pair, but these two nucleic acid fragments are complementary as defined herein. Typically two nucleic acid fragments are complementary if they hybridize under certain conditions.
As used herein, xe2x80x9chybridizes,xe2x80x9d xe2x80x9chybridizing,xe2x80x9d and xe2x80x9chybridizationxe2x80x9d means that a single stranded nucleic acid fragment forms a noncovalent interaction with a complementary nucleic acid fragment under certain conditions, as described herein.
As used herein, the term xe2x80x9cisolatedxe2x80x9d means that a nucleic acid fragment or polypeptide is either removed from its natural environment or synthetically derived. Preferably, the nucleic acid fragment or polypeptide is purified, i.e., essentially free from any other nucleic acid fragment or polypeptide and associated cellular products or other impurities.
xe2x80x9cS-triazines,xe2x80x9d xe2x80x9cs-triazine containing compoundsxe2x80x9d and xe2x80x9cs-triazine herbicidesxe2x80x9d are used interchangeably and refer to compounds and herbicides containing, for example, atrazine (2-chloro-4-ethylamino-6-isopropylamino- 1,3,5-s-triazine), desethylatrazine (2-chloro-4-amino-6-isopropylamino-s-triazine), deisopropylatrazine (2-chloro-4-ethylamino-6-amino-s-triazine), desethylhydroxyatrazine (2-hydroxy-4-amino-6-isopropylamino-s-triazine), desisopropylhydroxyatrazine (2-hydroxy-4-amino-6-isopropylamino-s-triazine), desethyldesisopropylatrazine (2-chloro-4,6-diamino-s-triazine), simazine (2-chloro-4,6-diethylamino-s-triazine), terbuthylazine (2-chloro-4-ethylamino-6-terbutylamino-s-triazine), melamine (2,4,6-triamino-s-triazine), ammelide (2,4-dihydroxy-6-amino-s-triazine), ammeline (2-hydroxy-4,6,-diamino-s-triazine), prometrym (N,Nxe2x80x2-bis(1-methylethyl)-6-(methylthio)-1,3,5-triazine-2,4 diamine), ametryn (N-ethyl-Nxe2x80x2-(1-methylethyl)-6-(methylthio)-1,3,5-triazine-2,4 diamine), and propazine (6-chloro-N,Nxe2x80x2-bis(1-methylethyl)-1,3,5-triazine-2,4-diamine).
The phenotypes of xe2x80x9cresistance,xe2x80x9d xe2x80x9cherbicide resistant,xe2x80x9d and xe2x80x9cherbicide tolerantxe2x80x9d refer to the ability of a cell or plant to survive or continue to grow in the presence of certain concentrations of an s-triazine that typically kill or inhibit the growth of other cells or plants. Growth includes, for instance, photosynthesis, increased rooting, increased height, increased mass, and development of new leaves. Although there may be other mechanisms of providing resistance, the plants of the present invention degrade s-triazines. Preferably, degradation of an s-triazine causes an s-triazine to be detoxified, such that it is less herbicidal for plants.
xe2x80x9cDegradationxe2x80x9d of an s-triazine includes, for instance, removing or changing a portion of the molecule, such as opening the ring structure. Degradation of an s-triazine can result in a compound having increased toxicity to a plant, a compound having about the same toxicity to a plant as the nondegraded s-triazine, or a compound having lower toxicity to a plant. A degraded s-triazine having lower toxicity is referred to herein as detoxified.
xe2x80x9cTransgenexe2x80x9d as used herein refers to an exogenous coding region present in a host cell. A transgene is preferably transmitted to progeny cells.
xe2x80x9cTransgenicxe2x80x9d as used herein refers to any cell, cell line, tissue plant part or plant the genotype of which has been altered by the presence of an exogenous coding region. Typically, the exogenous coding region was introduced into the genotype by a process of genetic engineering, or was introduced into the genotype of a parent cell or plant by such a process and is subsequently transferred to later generations by sexual crosses or asexual propagation.
A xe2x80x9cselectable markerxe2x80x9d or xe2x80x9cscreenable markerxe2x80x9d is a molecule that imparts a distinct phenotype to cells expressing the nucleic acid fragment encoding the marker and thus allow such transformed cells to be distinguished from cells that do not have the marker. A selectable marker confers a trait which one can xe2x80x98selectxe2x80x99 for by chemical means, i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like). A screenable marker confers a trait which one can identify through observation or testing, i.e., by xe2x80x98screeningxe2x80x99.
xe2x80x9cHybridxe2x80x9d refers to progeny plants resulting from a cross between parental lines.
xe2x80x9cInbredxe2x80x9d refers to progeny plants that are genetically homogeneous (homozygous) resulting from many generations of self crossing.
xe2x80x9cWaterxe2x80x9d as used herein refers to surface water, subsurface water, and ground water. The terms surface water, subsurface water, and ground water are described in greater detail herein.